Abstract

Cell division in Escherichia coli begins with assembly of the tubulin-like FtsZ protein into a ring structure just underneath the cell membrane. Spatial control over Z ring assembly is achieved by two partially redundant negative regulatory systems, the Min system and nucleoid occlusion (NO), which cooperate to position the division site at midcell. In contrast to the well-studied Min system, almost nothing is known about how Z ring assembly is blocked in the vicinity of nucleoids to effect NO. Reasoning that Min function might become essential in cells impaired for NO, we screened for mutations synthetically lethal with a defective Min system (slm mutants). By using this approach, we identified SlmA (Ttk) as the first NO factor in E. coli. Our combined genetic, cytological, and biochemical results suggest that SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid.

(A–D) DIC micrographs showing cells of TB43/pTB8 [ΔlaclZYA::frtΔminCDE::frt/Plao::minCDE::JacZ] (A and B) and its slmA127 derivative (C and D). Cells were grown to OD600 = 0.6–0.7 in liquid LB-Amp with (A and C) or without (B and D) 500 µM IPTG and fixed. Bar = 5 µm.(E) Diagram of the slmA locus indicating the location and orientation of the slmA127 and slmA267 EZTnKan-2 insertions (black triangles, inserted after bp 313 or 88 of slmA, respectively). SwissProt annotation P06969 was used to number amino acid residues. Potential DNA binding (HTH) and coiled-coil (CC) regions are indicated. The latter was identified by using COILS (). The double-headed arrow indicates the endpoints of the ΔslmA::aph and ΔslmA::frt deletion-replacement alleles.(F) Overnight cultures of TB28 [wt], TB57 [Para::minCDE], TB66 [slmA127], and TB68 [slmA127 Para::minCDE] were grown to equal density in LB-0.2% arabinose and serially diluted (10−1-10–6) in LB. Aliquots (5 µl) were spotted on LB agar without arabinose and incubated overnight at 30°C.

Cells of TB104 [cI857 λPR::dnaA] (A) and TB105 [ΔslmA::frtcI857 λPR::dnaA] (B and C) were grown in LB for 3.5 hr at 30°C to deplete DnaA. Cells were fixed, stained with DAPI, and imaged with DAPI- and DIC-specific optics. A1, B1, and C1 show a digital overlay of the DIC and DAPI images, and A2, B2, and C2 show the DIC image only. Bar = 2 µm. Several parameters of randomly selected cells from each strain were measured, and the results are summarized in (D).

DNA bound SlmA, presumed to be a dimer, competes with membrane bound septal ring components, such as ZipA and FtsA, for binding FtsZ polymers. In subsequent steps, SlmA may play a rather passive role in that SlmA bound polymers may simply turn-over due to their intrinsic GTP hydrolytic activity. A more attractive possibility is that SlmA, perhaps in combination with some other factor (X), actively promotes the disassembly of the FtsZ polymers.